Peptide-based carrier devices for stellate cells

ABSTRACT

A compound includes a carrier molecule wherein the carrier molecule is linked to a further molecule, wherein the further molecule is at least one cyclic peptide in which the cyclic peptide portion thereof contains at least one sequence encoding a cell receptor recognizing peptide (RRP) and with the proviso that the compound is not a naturally occurring receptor agonist or antagonist. Preferably, the RRP is a receptor specific for Hepatic Stellate Cells (HSC) or a receptor that is up-regulated on HSC during disease. The RFP may be chosen from among a PDGF receptor, a collagen type VI receptor, cytokine receptor(s) such as TGBβ, INFα and interleukinβ. The cyclic portion of the peptide can contain at least one amino acid sequence RGD or KPT. The compounds can be used as an active targeting ingredient for manufacturing a pharmaceutical composition for therapy, prophylaxis or diagnosis of a disease chosen from fibrotic disease, sclerotic disease, and chronic or acute inflammatory processes including glomeruloscherosis, interstitial fibrosis, lung fibrosis, atherosclerosis, rheumatoid arthritis, Crohns disease, colitis ulcerosa, glomerulonephritis and sepsis, and particularly for targeting HSC. Pharmaceutical compositions contain the above-compound(s).

PEPTIDE-BASED CARRIER DEVICES FOR STELLATE CELLS RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 11/012,056, filed Dec.15, 2004, which is a divisional of application Ser. No. 09/806,837,filed Jul. 23, 2001, now U.S. Pat. No. 6,344,319, issued Feb. 5, 2002,which is a U.S. National Phase of International ApplicationPCT/NL98/00579, filed Oct. 8, 1998, the entire disclosures of which,including any sequence listings, are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The inventions relate to methods for targeting cells involved insclerotic and/or fibrotic diseases in a tissue sample or subject and acomposition adapted for use in the disclosed methods.

BACKGROUND OF THE INVENTION

The hallmark of fibrosis is the excessive deposit of extracellularmatrix components caused by an increased synthesis and decreaseddegradation of matrix proteins, predominantly collagen type I and III.This process of fibrosis can occur in all kinds of organs such as thekidney (glomerulosclerosis or interstitial fibrosis), the skin (scarformation), the lung and also in the liver, where the end-stage of liverfibrosis is referred to as “cirrhosis.” The process also shares manycharacteristics with the formation of atherosclerotic plaques inarteries. Liver fibrosis leads to a deterioration of liver function, andeventually in complete liver failure, which is lethal if untreated. Theprocess can be elicited by viruses (Hepatitis A, B and C), alcoholconsumption, genetic disorders, or by chronic exposure to hepatotoxicagents. The incidence of this disease is very variable depending on thecountry. In the period 1985-1989, the incidence of liver cirrhosis inThe Netherlands was 3.90 per 100,000 habitants per year, whereas thisincidence in, for instance, France and Germany was respectively 11.9 and12.4. To date, no effective pharmacotherapeutic intervention isavailable for this disease. In the past decades, liver transplantationhas become a serious option for many patients, but the costs,availability of donor livers and traumatic nature of the transplantationprocedure itself hamper the application of such an operation in generalpractice. Pharmacological intervention would be a better option.

Hepatic stellate cells (HSC), also called “Ito cells” or “fat-storingcells” strongly proliferate during the progression of the disease, andthey subsequently transform into myofibroblasts (MF). These cells arethe major producers of collagens, glycoproteins, and proteoglycans in adiseased liver. Moreover, HSC and MF produce an array of mediators,which activate other hepatic and inflammatory cells, thus enhancing thefibrotic process. Therefore, HSC are an important target foranti-fibrotic therapy. However, in vivo studies indicate thatanti-fibrotic drugs are not efficiently taken up by HSC; and, as aconsequence, most drugs which show potent anti-fibrotic activity invitro, have failed to exert any effect in vivo. At high doses, suchdrugs often induce many side effects caused by extrahepatic distributionof the drug. Cell-specific delivery is an option to solve theseproblems. This can be accomplished by coupling drugs to carriermolecules which are selectively taken up by the target cells. Liposomesare well-known drug carriers, but modified proteins can also be applied.Cell-specific delivery of therapeutic and diagnostic agents tohepatocytes, endothelial and Kupffer cells has already been achieved bymodification of the sugar moieties of proteins or polymers. Coupling ofgalactose to, for instance, human serum albumin (HSA) leads to aspecific accumulation of this neoglycoprotein in hepatocytes, whereasthe addition of mannose to albumin causes uptake into Kupffer orendothelial cells. Increasing the net negative charge (for instance, bysuccinylation of amine groups) results in uptake of the protein intoendothelial cells via scavenger receptors. For a comprehensive review ofcarrier devices for cell-specific delivery of drugs, see D. K. F. Meijerand G. Molema, .Sem. in Liver Dis. 15: 202-256, 1995. The benefits ofsuch carrier devices on the development of novel pharmacotherapeuticinterventions are well recognized for various diseases. However, aspecific carrier for drugs to HSC, the most important cell in thepathogenesis of liver fibrosis, has not been found yet.

SUMMARY OF THE INVENTION

The invention describes novel drug carriers which specificallyaccumulate in hepatic stellate cells (HSC). These carriers can be usedfor the targeting of all kinds of therapeutic agents, preferablyanti-fibrotic drugs to HSC. The carriers may also be applied for thevisualization of HSC for diagnostic purposes. The basis of the inventionlies in the coupling of small cyclic proteins (oligopeptides) thatcontain specific receptor recognising peptides (RRPs) to soluble orparticle-type carriers (core carriers). The use of such a conjugate as atool for targeting purposes has not been described. The target-receptorsfor these neo proteins, neo oligopeptides or oligopeptide carrierconstructs are specific for HSC or are upregulated upon this cell typeduring the course of the disease. In the present study, human serumalbumin (HSA) is applied as the core-carrier, but the invention is notrestricted to a specific protein or polymer. Each molecule withattachment sites for peptides is applicable as a carrier to HSC. Theinvention describes conjugates which bind to the platelet-derived growthfactor (PDGF)-receptor and conjugates which attach to the collagen-typeVI receptor. Both types of receptors are present in relatively highamounts upon HSC and are well characterized. The respectivereceptor-binding ligands are known.

Since these receptors are also upregulated in renal mesangial cells, aswell as fibroblasts in various organs during glomerulosclerosis,interstitial fibrosis, lung fibrosis or atherosclerosis, and since thesepathological processes are accessible for macromolecules, it is assumedthat these carriers will also show a relative accumulation in these celltypes during the course of these diseases. The conjugates described heremay, therefore, also be applied as drug-carriers or carriers fordiagnostic markers and/or for treatment of the above-mentioned diseases.

The proliferation of HSC during the process of fibrosis is an importantpathogenic factor. Cell-matrix interactions and the production of growthfactors, such as PDGF, play a pivotal role in this proliferativeresponse of HSC. Peptides which bind to the PDGF receptors or collagentype VI receptors will block the binding of endogenous PDGF or willinterfere with cell-matrix interactions. Therefore, the oligopeptidesdescribed here may also exert an antiproliferative activity; and,consequently, these oligopeptides may serve as anti-fibrotic oranti-sclerotic agents themselves. Also, other receptors may be targetedusing this new approach. Transforming Growth Factor .beta. (TGF.beta.),interleukin 1.beta. (II 1.beta.), and Tumor Necrosis Factor .alpha.(TNF.alpha.) are other important mediators during chronic inflammatoryprocesses and the receptors for these cytokines are upregulated upon HSCas well as upon endothelial cells and Kupffer cells in the liver. Theligands for these receptors are well characterized and similar to thePDGF-receptor or collagen VI-receptor recognizing proteins, cyclicpeptides recognizing the binding site for these receptors can beprepared and coupled to a core-protein such as albumin. A relativeaccumulation of these conjugates can be expected into the target cellexpressing the particular receptors. Most of the cytokines contain a RGDsequence (arg-gly-asp). This is the (putative) cell attachment site andin combination with additional amino acids it will determine thespecificity for the individual cytokines and growth hormone receptors.Coupling this RGD sequence and accompanying amino acids to a carriermolecule using the approach described here is feasible. The inventionalso includes the preparation of oligopeptides in which more than onereceptor recognizing domain for the same receptor are combined andpeptide constructs in which different receptor recognizing domains fordifferent types of receptors are combined. The particular oligopeptideconstructs containing a single or more than one receptor recognizingdomain can be used as such, as intrinsic active substances but also forthe preparation of drug conjugates (pro-drugs) and be employed toprepare larger drug carriers by coupling of the oligopeptides to eitherproteins, soluble and particulate polymeric carriers and lipoid carriers(liposomes, neolipoproteins, micelles) that subsequently can be used forcovalent binding and/or inclusion or association of therapeutic agentsfor the purpose of cell-specific drug targeting. The application of suchcarriers is not limited to the treatment or diagnosis of fibroticprocesses but also to other chronic and acute inflammatory processessuch as, for instance, rheumatoid arthritis, Crohn's disease, colitisulcerosa, glomerulonephritis and sepsis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-a shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing thePDGF-receptor in normal rats.

FIG. 1-b shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing thePDGF-receptor in rats with liver fibrosis induced by bile duct ligation(3 weeks after the operation).

FIG. 1-c represents the organ distribution of unmodified HSA.

FIG. 2-a shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing the collagentype VI-receptor in normal rats.

FIG. 2-b shows organ distribution of human serum albumin (HSA)conjugated with 10 to 12 cyclic oligopeptides recognizing the collagentype VI-receptor in rats with liver fibrosis induced by bile ductligation (3 weeks after the operation).

FIG. 3-a shows that after intravenous administration of modifiedprotein, the albumin derivatives can be immunohistochemically detectedin a non-parenchymal cell type of the liver using a polyclonal antibodyagainst albumin.

FIG. 3-b shows, as seen from the arrowheads, the modified albuminco-localizes with the marker for HSC (desmin).

FIG. 4 is a graph representing in vitro displacement of radiolabeledPDGF-BB from its receptor upon 3T3-fibroblasts by HSA-PDGFreceptor-binding peptide conjugates (pPB-HSA, closed blocks), HSA (openblocks) or uncoupled PDGF-receptor binding peptides (pPB, open circles).

FIG. 5 is a graph of the organ distribution of radiolabeled M6P^(x)-HSAin fibrotic rats (three weeks after bile duct ligation), 10 minutesafter intravenous administration of the modified HSA.

FIG. 6 is a graph of the binding and uptake of radiolabeled M6P₂₈-HSA inhuman liver tissue at the reported temperatures.

Suitably, when the RRP is of a collagen-type VI receptor, cytokinereceptor such as TGFβ, TNFα and interleukin 1β, the cyclic portion ofthe cyclic peptide comprises at least the amino acid sequence RGD or KPT(lys-pro-thr) in the cyclic portion thereof. By way of example, thecyclic portion of the cyclic peptide comprises at least an amino acidsequence selected from X*YRGDYX*(Xaa(Xaa)_(n)-arg-gly-asp-(Xaa)_(n)-Xaa) and X*YKPTYX*(Xaa-(Xaa)_(n)-lys-pro-thr-(Xaa)_(n)-Xaa) wherein X* represents thelocation of cyclisation and Y represents at least one amino acid or asequence of amino acids up to a length such that the receptor bindingcapacity of the cyclic peptide is retained. In a preferred embodiment,X* represents the location of attachment to the carrier molecule. In anembodiment illustrating the above, when the receptor agonist is of acollagen type VI receptor has a cyclic portion of the cyclic peptidecomprising the amino acid sequence X*GRGDSPX*(Xaa-gly-arg-gly-asp-ser-pro-Xaa). Suitably, it will comprise thesequence -cysteine-glycine-arginine-glycine-asparticacid-serine-proline-cysteine. SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

A compound according to the invention comprises a carrier molecule, saidcarrier molecule being linked to a further molecule, said furthermolecule being at least one cyclic peptide, said cyclic peptidecomprising in the cyclic peptide portion thereof at least one sequencecontaining at least one specific receptor recognising peptide (RRP) andwith the proviso the compound is not a naturally occurring receptoragonist or antagonist. Suitably in such a compound according to theinvention the RRP is of a receptor specific for Hepatic Stellate Cells(HSC) or a receptor that is upregulated on HSC during disease.

The RRP can by way of example be the agonist or antagonist of a receptorselected from the group of receptors consisting of PDGF receptor,collagen type VI receptor, cytokine receptor such as TGF.beta.,TNF.alpha. and interleukin 1.beta.

Suitably, when the RRP is of a collagen type VI receptor, cytokinereceptor such as TGFβ, TNFα and interleukin 1β, the cyclic portion ofthe cyclic peptide comprises at least the amino acid sequence RGD or KPT(lys-pro-thr) in the cyclic portion thereof. By way of example, thecyclic portion of the cyclic peptide comprises at least an amino acidsequence selected from X*YRGDYX*(Xaa(Xaa)_(n)-arg-gly-asp-(Xaa)_(n)-Xaa) and X*YKPTYX*(Xaa-(Xaa)_(n)-lys-pro-thr-(Xaa)_(n)-Xaa) wherein X* represents thelocation of cyclisation and Y represents at least one amino acid or asequence of amino acids up to a length such that the receptor bindingcapacity of the cyclic peptide is retained. In an preferred embodiment,X* represents the location of attachment to the carrier molecule. In anembodiment illustrating the above, when the receptor agonist is of acollagen type VI receptor has a cyclic portion of the cyclic peptidecomprising the amino acid sequence X*GRGDSPX*(Xaa-gly-arg-gly-asp-ser-pro-Xaa). Suitably, it will comprise thesequence -cysteine-glycine-arginine-glycine-asparticacid-serine-proline-cysteine. SEQ ID NO:1.

Suitably, when the receptor agonist is of an interleukin 1 betareceptor, the cyclic peptide can comprise the amino acid sequenceX*DKPTLX* (Xaa-asp-lys-pro-thr-lys-Xaa). SEQ ID NO:2.

Alternatively, when the receptor agonist is of PDGF receptor, the cyclicportion of the cyclid peptide can comprise the amino acid sequenceX*SRNLIDCX* (Xaa-ser-arg-asn-leu-ile-asp-cys-Xaa), wherein X* representsthe location of cyclisation. SEQ ID NO:3. In a preferred embodiment, X*represents the location of attachment to the carrier molecule. Such acompound will bind to the PDGF receptor alha and beta subtypes.Suitably, it will comprise the sequence-cysteine-serine-arginine-asparagine-leucine-isoleucine-asparticacid-cysteine.

In any of the embodiments according to the invention that are described,X* can be a cysteine residue.

Only some crucial amino acids for the cyclic peptides are provided here.The oligopeptide may be elongated without causing a change in thecellular distribution pattern in vivo. Cyclisation of these peptides canbe achieved, for example, by a disulfide bond between both cysteinegroups. The free amine (a-amine) in one cysteine residue can be used tocouple the oligopeptide to the carrier molecule. For example to theamine groups in a core-molecule like albumin, using succinimide-acetylthioacetate (SATA). Coupling of more than one oligopeptide to albumincan be readily done. Attachment of the cyclic peptides to a carriermolecule via a biodegradable spacer, causing local release of the cyclicpeptides, is also feasible. The examples provided here describeconjugates with multiple oligopeptides per HSA molecule, leaving enoughfree reactive groups within the core-protein (hydroxyl, amine orsulphate groups) to attach additional drugs or other chemicals. Theseconjugates selectively accumulate in HSC of normal and diseased livers.

The cyclic portion of the cyclic peptide can suitably comprise multipleRRP sequences. The cyclic portion of the cyclic peptide can comprisemultiple RRP sequences directed at least two different types ofreceptors. Obviously they can also be directed at the same type ofreceptor. Combinations of various receptor agonist sequences arenaturally also possible. Thus a compound according to the invention inany of the embodiments defined may comprise multiple cyclic peptidesdirected at the same or different subtypes of receptors or may comprisemultiple but similar oligopeptides that contain more than one identicalor different RRP sequence directed at the same receptor or differentreceptors on the particular cell type respectively. By way of example, acompound according to the invention, wherein the carrier molecule islinked to more than one cyclic peptide can suitably comprise 5-15 cyclicpeptides as defined in any of the embodiments above.

A person skilled in the art will realise that numerous types of carriermolecules can be applied. The carrier molecule can suitably be selectedfrom endogenous plasma proteins, e.g., albumin, lactoferrin, alkalinephosphatase, superoxide dismutase, alpha2 macroglobulin and fibronectin.They are to be pharmaceutically acceptable and of a size such that theypreferably are not lost due to the renal excretion thereof. Suchcompounds are suitably larger than 5000 Daltons. Suitable examples ofthe carrier molecule can be selected from the group of carrier moleculesconsisting of proteins, oligo or polypeptides, immunoglobulins or partsthereof, oligonucleotides, disaccharides, polysaccharides, biodegradablesynthetic polymers, liposomes, lipid particles, biocompatible polymersin the form of microspheres or nanoparticles. Quite suitably, in acompound according to this aspect of the invention, the carrier moleculeis the endogenous plasma protein albumin. The immunoglobulins can bemono or polyclonal. Parts of immunoglobulins can comprise Fab′ fragmentsor single chain Ig. Humanised antibodies and bispecific antibodies areenvisaged. In the case of human administration, carriers that occurnaturally in humans are preferred. For the sake of easy linkage of thecarrier molecule to the cyclic peptide, the carrier molecule preferablycomprises free reactive groups such as hydroxyl, amine or sulphate. Thecarrier molecule can suitably be linked to the cyclic peptide via abiodegradable spacer. The carrier molecule can itself be a drug, theactivity of which is not impaired by linking the cyclic peptide to it.

In an alternative embodiment of the invention, the carrier molecule inthe compound can comprise additional drugs or chemicals linked thereto.

The invention also covers a pharmaceutical composition comprising acompound according to any of the aforementioned embodiments as thetargeting ingredient and any pharmaceutically acceptable carrier. Apharmaceutical composition according to the invention comprises acompound in any of the embodiments mentioned above as pharmaceuticallythe active ingredient in combination with any pharmaceuticallyacceptable additional carrier. In an alternative embodiment, thepharmaceutical composition can further comprise a drug biodegradablyattached to the compound. It is also possible for the compound tofurther comprise a diagnostic marker attached thereto. A pharmaceuticalcomposition according the invention will be in a pharmaceutical dosageform. Such a dosage form can comprise sprayable, injectable or infusablesolutions or solids or dosage forms for pulmonary or otheradministration routes. Also, a pharmaceutical composition according tothe invention can be in a topical form. In the case of parenteraladministration, a systemically acceptable form should be composed. Thismeans it can enter the bloodstream without causing clotting orinadmissibly toxic reactions.

The invention is also directed at application of a compound according tothe invention in any of the above-mentioned embodiments as the activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the invention for therapy, prophylaxis or diagnosis ofchronic diseases. Examples from this group consist of fibrotic disease,sclerotic disease and chronic or acute inflammatory processes such asglomerulosclerosis, interstitial fibrosis, atherosclerosis, rheumatoidarthritis, Crohns disease, colitis ulcerosa, glomerulonephritis, lungfibrosis and sepsis. Suitable use of a compound according to theinvention as the active targeting ingredient for manufacturing apharmaceutical composition according to the invention for therapy,prophylaxis or diagnosis of a disease related to proliferation of HSC isalso envisaged as forming a particularly useful application to becovered by the invention. A method of targeting HSC, said methodcomprising administration in a pharmaceutically acceptable amount andform of a compound or a pharmaceutical composition according to theinvention to a subject or a tissue sample of a subject is covered by theinvention. The person skilled in the art will adjust the dosage to beapplied to the manner of application, size, weight, state of health,etc. of the subject to which administration is to occur. Administrationcan occur in any manner known per se for the administration ofmedicament.

The invention also covers a method of therapy, diagnosis or prophylaxisof a disease related to HSC, said method comprising administration in apharmaceutically acceptable amount and form of a compound or apharmaceutical composition according to the invention to a subject or atissue sample of a subject. In particular, such disease can be oneselected from the group consisting of fibrotic disease, scleroticdisease and chronic or acute inflammatory processes such asglomerulosclerosis, interstitial fibrosis, lung fibrosis,atherosclerosis, rheumatoid arthritis, Crohns disease, colitis ulcerosa,glomerulonephritis and sepsis. The method comprises administration in apharmaceutically acceptable amount and form of the compound orpharmaceutical composition according to the invention to a subject or atissue sample of a subject. The person skilled in the art will adjustthe dosage to be applied to the manner of application, size, weight,state of health, etc. of the subject to which administration is tooccur. Administration can occur in any manner known per se for theadministration of therapeutic agents.

This further aspect of the invention is illustrated in, but not limitedto, the following examples.

EXAMPLE 1

Normal rats and rats with liver fibrosis (3 weeks after bile ductligation) received an intravenous injection of 10 mg/kg b.w. PDGFreceptor-binding peptides conjugated with HSA. Based upon the results ofthe organ distribution studies with radiolabeled conjugates (FIG. 1),rats were sacrificed after ten minutes, and samples of the liver andbone (from ribs, front paw, rear paw and the back) were removed forhistochemical examination. No accumulation of this HSA-peptide conjugatewas detectable in bone samples, whereas abundant staining was found intissue samples. Upon cryostat sections of these livers double stainingswere performed with anti-HSA antibodies and antibodies against Kupffercells (ED1), endothelial cells (RECA-1), myofibroblasts (anti-actinantibodies) or hepatic stellate cells (desmin and GFAP antibodies).Subsequently, the number of double positive cells (HSA+ and cellmarker+) were counted and related to the total number of HSA positivecells in the same area. Results of the quantitative evaluation of thecarrier uptake in the liver are summarized in table 1.

TABLE 1 Relative accumulation of HSA modified with collagen VI-receptorrecognising peptides (pCVI-HSA) or PDGF receptor-recognising peptides(pPB-HSA) in non-parenchymal cells of the liver. The number ofHSA-positive cells was related to the number of cells double-positivefor HSA and a HSC marker (desmine), or a EC marker (HIS 52), or a KCmarker (ED2) or a PC marker (glycogen). % HSC % EC % KC PC pCVI-HSA 73 ±14 30 ± 10 16 ± 11 − pPB-HSA 72 ± 18 16 ± 6  11 ± 6  + HSC = hepaticstellate cells, EC = endothelial cells, KC = Kupffer cells, PC =parenchymal cells

EXAMPLE 2

Normal rats and rats with liver fibrosis (3 weeks after bile ductligation) received an intravenous injection of 10 mg/kg b.w. collagen VIreceptor-binding peptides conjugated with HSA. Based upon the results ofthe organ distribution studies with radiolabeled conjugates (FIG. 2),rats were sacrificed after ten minutes, and samples of the liver andbone (from ribs, front paw, rear paw and the back) were removed forhistochemical examination. No accumulation of this HSA-peptide conjugatewas detectable in bone samples, whereas abundant staining was found intissue samples. Upon cryostat sections of these livers, double stainingswere performed, with anti-HSA antibodies and antibodies against Kupffercells (ED1), endothelial cells (RECA-1), myofibroblasts (anti-actinantibodies) or hepatic stellate cells (desmin and GFAP antibodies).Subsequently, the number of double-positive cells (HSA+ and cellmarker+) were counted and related to the total number of HSA positivecells in the same area. Results of the quantitative evaluation of thecarrier uptake in the liver are summarized in table 1.

EXAMPLE 3

Another cyclic oligopeptide recognizing the PDGF-receptor can bedescribed as follows:

-   -   -cysteine-arginine-lysine-lysine-proline-cysteine-(C*RKKPC*),

where the cysteines (C*) represent the cyclisizing residues.

Only some crucial amino acids for the PDGF-binding peptide are providedhere. The oligopeptide may be elongated without causing a change in thecellular distribution pattern in vivo. Cyclisation of this peptide canbe achieved by a disulfide bond between both cysteine groups, whereasthe free amine in one cysteine residue can be used to couple theoligopeptide to a core-molecule like albumin. Coupling of more than oneoligopeptide to albumin can be readily done.

EXAMPLE 4

A cyclic peptide which binds to the interleukin 1′-receptor can bedescribed as follows:

-cysteine-asparticacid-lysine-proline-threonine-leucine-cysteine-(-C*DKPTLC*)

where the cysteines (C*) represent the cyclisizing residues.

The receptor binding properties of the tripeptidelysine-proline-threonine (KPT) has been reported (Ferreira et al.,Nature 334:698, 1988). Two or more additional amino acids, preferablythe two adjacent amino acids in the native interleukin 1.beta. moleculeon both sites of this tripeptide, are in this example attached to thistripeptide. Subsequently, the terminal cysteine residues allow forcyclysation of this oligopeptide and coupling of this peptide to amacromolecule. In this way, the interleukin 1.beta. binding site isexposed to its receptor similar to the PGDF— and collagen VI receptorbinding peptides. This conjugate may also serve as a carrier fortherapeutic or diagnostic agents for the treatment of inflammatoryprocesses.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Organ distribution of human serum albumin (HSA) conjugated with10 to 12 cyclic oligopeptides recognizing the PDGF-receptor in normalrats [FIG. A] and in rats with liver fibrosis induced by bile ductligation (3 weeks after the operation) [FIG. B]. FIG. C represents theorgan distribution of unmodified HSA. Organs were removed 10 minutesafter intravenous administration of radiolabeled (.sup.125I) protein andanalyzed using a gamma-counter. The results are expressed as themean.+−.SD (n=3 per group). Note the accumulation of modified HSA inlivers of normal and diseased rats, whereas native HSA remains in theblood.

FIG. 2: Organ distribution of human serum albumin (HSA) conjugated with10 to 12 cyclic oligopeptides recognizing the collagen type VI-receptorin normal rats [FIG. A] and in rats with liver fibrosis induced by bileduct ligation (3 weeks after the operation) [FIG. B]. Organs wereremoved 10 minutes after intravenous administration of radiolabeled(.sup. 125I) protein and analyzed using a gamma-counter. The results areexpressed as the mean.+−. SD (n=3 per group). Note the accumulation ofmodified HSA in livers of normal and diseased rats.

FIG. 3: Intrahepatic distribution of HSA modified with 10-12 collagentype VI-receptor binding peptides in fibrotic rats (3 weeks after bileduct ligation). 10 minutes after intravenous administration of modifiedprotein, the albumin derivatives can be immunohistochemically detectedin a non-parenchymal cell type of the liver using a polyclonal antibodyagainst albumin [FIG. A]. The modified albumin co-localizes with themarker for HSC (desmin) [see arrowheads, FIG. B].

FIG. 4: In vitro displacement of radiolabeled PDGF-BB from its receptorupon 3T3-fibroblasts by HSA-PDGF receptor-binding peptide conjugates(pPB-HSA, closed blocks), HSA (open blocks) or uncoupled PDGF-receptorbinding peptides (pPB, open circles). Note the strong inhibition ofbinding of native PDGF to fibroblasts induced by the modified HSA, butnot by native HSA or the oligopeptides alone.

BACKGROUND INFORMATION TO A FURTHER ASPECT OF THE INVENTION

An increased expression of the Insulin Growth Factor11/mannose-6-phosphate (IGFII/M6P) receptor has been reported uponhepatic stellate cells in particular after activation of this cell type.This led to the idea of coupling mannose-6-phosphate (M6P) to acore-protein and the use of such a neo-glycoprotein as a drug carrier toHSC. The degree of substitution of M6P to HSA necessary for this purposecould not be deduced from the present state of the art. We have foundthat a quite high degree of substitution is required for efficienttargeting. Introduction of only a few groups was not particularlysuccessful. The invention thus provides a novel type of drug carrier tothe hepatic stellate cells (HSC). The carrier can be used for thetargeting of all kinds of therapeutic agents, preferably anti-fibroticagents to HSC, or may be applied for the visualization of HSC fordiagnostic purposes.

As it was also reported that this receptor played a role in theactivation of latent TGF-beta and TGF-beta is known to be apro-fibrogenic growth factor which is a very important mediator duringfibrosis the compound according to this further aspect of the inventionshould also be useful for diagnosis, prophylaxis and therapy of fibroticdiseases. Mannose-6-phosphate substituted proteins may also interferewith the activation of latent TGF-beta and this carrier may thereforehave an antifibrotic action of its own.

DETAILED DESCRIPTION OF THE FURTHER ASPECT OF THE INVENTION

The invention in a further aspect is directed at a compound capable ofrecognising and binding a mannose 6 phosphate receptor said compoundcomprising a carrier molecule linked to a molecule capable ofrecognising and capable of binding mannose-6-phosphate receptor, saidmolecules recognising and capable of binding mannose-6-phosphatereceptor being present on the carrier molecule in at least an amountsufficient to occupy at least 20% of the carrier molecule linking sitesfor said molecules recognising and capable of bindingmannose-6-phosphate receptor, with the proviso the compound is notlatent tumor growth factor beta, thyroglobulin or a lysosomal protein.The latter are known proteins that are also known to comprise terminalmannose 6 phosphate groups and as such will bind to the mannose 6phosphate receptor. They are excluded as compounds according to theinvention. The substitution degree can be higher than 30% even as highas 40 or 50%. A suitable example of the molecule capable of recognisingand capable of binding mannose-6-phosphate receptor is mannose 6phosphate.

In a compound according to this aspect of the invention the carriermolecule can be selected from the group consisting of proteins, oligo orpolypeptides, immunoglobulins or parts thereof, oligonucleotides,disaccharides, polysaccharides, biodegradable synthetic polymers,liposomes, lipid particles, biocompatible polymers in the form ofmicrospheres or nanoparticles. The carrier molecule can suitably beselected from endogenous plasma proteins, e.g., albumin, lactoferrin,alkaline phosphatase, superoxide dismutase, alpha2 macroglobulin andfibronectin. The immunoglobulins can be mono or polyclonal. Parts ofimmunoglobulins can comprise Fab′ fragments or single chain Ig.Humanised antibodies and bispecific antibodies are envisaged. Quitesuitably in a compound according to this aspect of the invention thecarrier molecule is the endogenous plasma protein albumin. A personskilled in the art will realise numerous types of carrier molecules canbe applied. They are to be pharmaceutically acceptable and of a sizesuch that they preferably are not lost due to the renal excretionthereof. Such compounds are suitably larger than 50000 Daltons.

Quite specifically in a preferred embodiment of a compound according tothis aspect of the invention at least 10 molecules capable ofrecognising and capable of binding mannose-6-phosphate receptor arelinked to the carrier molecule. The carrier based upon macromoleculessubstituted with mannose-6-phosphate residues with substitution of morethan 10 mannose-6-phosphate residues per macromolecule has been foundexceptionally appropriate for proper targeting. The carrier molecule washuman serum albumin.

The invention also covers a pharmaceutical composition comprising acompound according to any of the aforementioned embodiments of thefurther aspect of the invention disclosed as targeting ingredient andany pharmaceutically acceptable carrier. A pharmaceutical compositionaccording to the invention comprises a compound in any of theembodiments mentioned above as pharmaceutically active ingredient incombination with any pharmaceutically acceptable additional carrier. Inan alternative embodiment the pharmaceutical composition can furthercomprise a drug biodegradably attached to the compound. It is alsopossible for the compound to further comprise a diagnostic markerattached thereto. A pharmaceutical composition according the inventionwill be in a medicinal dosage form. Such a dosage form can comprisesprayable, injectable or infusable solutions or solids or dosage formsfor pulmonary or other administration routes. Also a pharmaceuticalcomposition according to the invention can be in a topical form but willpreferably be in a systemically acceptable form. This means it can enterthe bloodstream without causing clotting or inadmissibly toxic reaction.

The invention is also directed at application of a compound according tothe invention in any of the abovementioned embodiments as activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the further aspect of the invention just mentioned fortherapy, prophylaxis or diagnosis of a disease selected from the groupof chronic diseases, for example fibrotic disease, sclerotic disease andchronic or acute inflammatory processes such as glomerulosclerosis,interstitial fibrosis, lung fibrosis, atherosclerosis, rheumatoidarthritis, Crohns disease, colitis ulcerosa, glomerulonephritis andsepsis.

The invention is also directed at application of a compound according tothe invention in any of the abovementioned embodiments as activetargeting ingredient for manufacturing a pharmaceutical compositionaccording to the further aspect of the invention just mentioned fortherapy, prophylaxis or diagnosis of any of the following pathologicalconditions; cell proliferation associated pathology e.g. tumors, adisease related to proliferation of HSC, fibroblast proliferationassociated pathology, endothelial cell proliferation associatedpathology and osteoblast proliferation associated pathology.

The invention also covers a method of targeting proliferating cells,preferably tumor cells, HSC, fibroblasts, endothelial cells andosteoblasts said method comprising administration in a pharmaceuticallyacceptable amount and form of a compound or a pharmaceutical compositionaccording to the aspect of the invention just described to a subject ora tissue sample of a subject.

In an alternative embodiment it also covers a method of targetingproliferating cells, preferably tumor cells, HSC, fibroblasts,endothelial cells and osteoblasts said method comprising administrationin a pharmaceutically acceptable amount and form of a compound or apharmaceutical composition according to the further aspect of theinvention just described to a subject or a tissue sample of a subject.Alternatively it covers a method of therapy, diagnosis or prophylaxis ofa disease related to proliferating cells, preferably tumor cells, HSC,fibroblasts, endothelial cells and osteoblasts, said method comprisingadministration in a pharmaceutically acceptable amount and form of acompound or a pharmaceutical composition according to the further aspectof the invention to a subject or a tissue sample of a subject.Specifically it covers a method of therapy, diagnosis or prophylaxis ofa disease related to HSC, said method comprising administration in apharmaceutically acceptable amount and form of a compound or apharmaceutical composition as described for the further aspect of theinvention to a subject or a tissue sample of a subject. A method oftherapy, prophylaxis or diagnosis of a disease selected from the groupconsisting of fibrotic disease, sclerotic disease and chronic or acuteinflammatory processes such as glomerulosclerosis, interstitialfibrosis, lung fibrosis, atherosclerosis, rheumatoid arthritis, Crohnsdisease, colitis ulcerosa, glomerulonephritis and sepsis, said methodcomprising administration in a pharmaceutically acceptable amount andform of a compound or a pharmaceutical composition as described for thefurther aspect of the invention to a subject or a tissue sample of asubject also falls within the scope of the invention.

This further aspect of the invention will be illustrated but not limitedin the following examples.

EXAMPLE 5

Mannose 6-phosphate was covalently coupled to human serum albumin (HSA)in two steps. First, p-nitrophenyl-.alpha.-D-mannopyranoside (Sigma, St.Louis, USA) was phosphorylated according to standard procedures. Themolecular weight (MW 381) and purity of the obtained crystalline productp-nitrophenyl-6-phospho-.alpha.-D-mannopyranoside was verified by massspectrometry. Subsequently, the nitro-group was reduced with 10%palladium on active carbon (Aldrich Chemie GmbH, Steinheim, Germany)under hydrogen atmosphere of 1 atm. The obtained productp-aminophenyl-6-phospho-.alpha.-D-mannopyranoside was coupled to HSA byactivation with thiophosgene. By variations in the molar ratio HSA:p-nitrophenyl-6-phospho-.alpha.-D-mannopyranoside, a series ofneoglycoproteins (M6P.sub.x-HSA) were obtained, x=2, 4, 10, 21, or 28.The M6P.sub.x-HSA preparations were further purified and characterizedaccording to standard procedures.

A tracer dose of modified HSA (.sup.125I labelled) was intravenouslyadministered to normal and fibrotic rats (three weeks after bile ductligation). Ten minutes after injection of these compounds, rats weresacrificed and all organs were removed. As can also be seen in FIG. 5,the degree of substitution of mannose 6-phosphate to HSA stronglyinfluenced liver uptake. HSA with a low degree of sugar loading (x=2-10)accumulated for 2. +−. 1% to 9. +−.0.5% in fibrotic rat livers, whilethe rest of the dose remained in the circulation. An increase in themolar ratio of M6P:HSA up to 28 caused a gradual increase in liveraccumulation (to 59. +−0.9% of the dose).

In addition, the intrahepatic distribution of modified HSA was examinedimmunohistochemically. Modified HSA was administered to rats (10 mg/kgb.w.) and 10 minutes after the injection samples from the liver, spleen,kidney, and bone were histochemically examined. We observed that themore mannose 6-phosphate was linked to HSA, the higher the uptake was inHSC. Quantitative evaluation of liver sections ten minutes afteradministration of modified HSA revealed that M6P10-HSA accumulated for19. +−. 10% in HSC. In contrast, 69.+−0.12% of the intrahepatic stainingfor M6P28-HSA was found in HSC, whereas 20.+−0.6% was found in Kupffercells and 17.+−0.6% in endothelial cells. No uptake was detected inhepatocytes and bile duct epithelial cells. Also no staining formodified HSA was found in other organs.

EXAMPLE 6

M6P₂₁-bovine serum albumin (BSA) and M6P₂₈-HSA, synthesized andcharacterized according to standard procedures, were radiolabeled with¹²⁵I. The intrahepatic uptake of these neo-glycoproteins was measured inhuman liver slices. These slices (+10 mg liver tissue with a thicknessof approximately 10 cells) were obtained from patients with normal liverfunction and from cirrhotic patients. Significant intrahepaticaccumulation of radiolabeled BSA and HSA derivates was found within onehour after co-incubation with these slices, whereas unmodified BSA orHSA was not taken up by the human tissue samples (see FIG. 6).

EXAMPLE 7

Pyrrolidine-dithiocarbamate (PDTC, which is an inhibitor of thetranscription factor NF-kappaB) was attached to M6P₂₈-HSA by couplingthe carboxylic groups of PDTC to lysine groups of HSA according tostandard procedures. This compound was administered to rats withliverfibrosis induced by bile duct ligation. Rats receiving thisconjugate 1, 3 and 5 days after the bile duct ligation displayed lessproliferation of HSC in the parenchymal area at day 7 as compared torats receiving no treatment or PDTC alone after induction of fibrosis.HSC were demonstrated in cryostat sections with anti-desmine andanti-Glial Fibrillar Acidic Protein (GFAP) antibodies and standardindirect immunoperoxidase techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 5: The organ distribution of radiolabeled M6P.sup.x-HSA in fibroticrats (three weeks after bile duct ligation), 10 minutes afterintravenous administration of the modified HSA. x=2, 4, 10, 21, and 28.Note that proteins substituted with 2, 4 or 10 M6P molecules per HSAremain in the blood, whereas proteins with high amounts of substitutionaccumulate in the liver.

FIG. 6. Binding and uptake of radiolabeled M6P.sub.28-HSA in human livertissue at 4 degrees Celcius respectively 37 degrees Celsius..sup.125I-labeled modified albumin was incubated with slices (10 mg)obtained from patients with a normal liver function. (TX=transplantationliver) or from patients with liver Cirrhosis (Cir).

Note the high accumulation of neo-glycoprotein in human liver slices ascompared to native HSA.

1. (canceled) 2-26. (canceled)
 27. A compound comprising a carriermolecule linked to at least one further molecule selected from the groupconsisting of: (a) a carrier molecule linked to at least 10 moleculescapable of recognising and capable of binding mannose-6-phosphatereceptor are linked to said carrier molecule, with the proviso thecompound is not a naturally occurring peptide with terminalmannose-6-phosphate residues, latent growth factor beta, thyroglobulinor lyosomal protein, (b) a carrier molecule linked to at least onefurther molecule which is X*YRGDYX* wherein X* represents the locationof cyclisation and Y represents at least one amino acid or a sequence ofamino acids up to a length such that the target receptor bindingcapacity of the further molecule is retained, (c) a carrier moleculelinked to at least one further molecule wherein said further molecule isX*YKPTYX*, wherein X* represents the location of cyclisation and Yrepresents at least one amino acid or a sequence of amino acids up to alength such that the target receptor binding capacity of the furthermolecule is retained, and (d) a carrier molecule linked to at least onefurther molecule wherein said at least one further molecule is X*RKKPX*,wherein X* represents the location of cyclisation.
 28. A compoundaccording to claim 27, wherein the molecule capable of recognising andcapable of binding to mannose-6-phosphate receptor ismannose-6-phosphate.
 29. A compound according to claim 27, wherein saidfurther molecule is X*GRGDSPX*, wherein X* represents the location ofcyclisation.
 30. A compound according to claim 27, wherein said furthermolecule is X*DKPTLX*, wherein X* represents the location ofcyclisation.
 31. A compound according to claim 27, wherein X* is acystein residue.
 32. A compound according to claim 27, wherein X*represents the location of cyclisation and attachment to the carriermolecule.
 33. A compound according to claim 27, wherein in the furthermolecule of (b)-(d), the cyclic portion of the cyclic peptide comprisesmultiple receptor binding sequences.
 34. A compound according to claim27, wherein of the further molecule the cyclic portion of the cyclicpeptide comprises multiple receptor binding sequences directed at leasttwo different types of receptors.
 35. A compound according to claim 27,wherein the further molecule in (b)-(d) comprises multiple cyclicpeptides directed at the same or different types of receptors.
 36. Acompound according to claim 27, wherein the carrier molecule is selectedfrom the group of carrier molecules consisting of proteins, oligo orpolypeptides, immunoglobulins or parts thereof, oligonucleotides,disaccharides, polysaccharides, biodegradable synthetic polymers,liposomes, lipid particles, biocompatible polymers in the form ofmicrospheres or nanoparticles, endogenous plasma proteins, lactoferrin,alkaline phosphatase, superoxide dismutase, alpha2 macroglobulin andfibronectin.
 37. A compound according to claim 27, wherein the carriermolecule comprises additional drugs or chemicals linked thereto.
 38. Acompound according to claim 27, wherein the carrier molecule comprises adiagnostic marker attached thereto.
 39. A compound according to claim27, wherein said compound is a targeting ingredient and is incombination with one or more pharmaceutically acceptable carriers.
 40. Amethod selected from the group consisting of: (a) a method comprisingusing a compound according to claim 27 in vitro diagnosis of a scleroticand/or fibrotic disease selected from the group consisting of liverfibrosis, kidney fibrosis, lung fibrosis, atherosclerosis and chronic oracute inflammatory processes Crohns disease, colitis ulcerosa,glomerulonephritis, sepsis and tumor-cell proliferation associatedpathology, fibroblast proliferation associated pathology, endothelialcell proliferation associated pathology and osteoblast proliferationassociated pathology; (b) a method comprising using a compound accordingto claim 27 for the preparation of a medicament for in vivo diagnosis,prophylaxis and/or therapy of a sclerotic and/or fibrotic diseaseselected from the group consisting of liver fibrosis, kidney fibrosis,lung fibrosis, atherosclerosis and chronic or acute inflammatoryprocesses, Crohns disease, colitis ulcerosa, glomerulonephritis, sepsisand tumor-cell proliferation associated pathology, fibroblastproliferation associated pathology, endothelial cell proliferationassociated pathology and osteoblast proliferation associated pathology.41. The method of claim 40, wherein said method is (a) a method for invitro diagnosis of a sclerotic and/or fibrotic disease, said methodcomprising the steps of providing a tissue sample of a subject andadministering a compound according to claim 31 to said tissue sample,wherein said disease is selected from the group consisting of liverfibrosis, kidney fibrosis, lung fibrosis, atherosclerosis and chronic oracute inflammatory processes, Crohns disease, colitis ulcerosa,glomerulonephritis, sepsis and tumor-cell proliferation associatedpathology, fibroblast proliferation associated pathology, endothelialcell proliferation associated pathology and osteoblast proliferationassociated pathology.